Project Summary/Abstract This application joins the efforts of two co-principal investigators, one a new and early stage investigator, an orthopaedic surgeon-scientist who recently completed a National Cancer Institute sponsored K08 career development award focused on mouse modeling of sarcomagenesis, the other an experienced biochemist with expertise in chromatin remodeling complexes and genomics. These investigators have assembled a team to bring their varied experience to bear on the core epigenetic mechanisms of synovial sarcomagenesis. Synovial sarcoma is the most common soft-tissue sarcoma of adolescence and young adulthood. It is driven by a single genetic aberration: the creation of a fusion oncogene from a balanced chromosomal translocation t(X;18). In models developed by the investigators and their colleagues, expression of these SS18-SSX fusion oncogenes in the mouse drives faithful recapitulations of human synovial sarcoma. The SS18-SSX fusion oncoproteins have been shown in human synovial sarcoma cell lines to utilize two epigenetic mechanisms to impact the transcription of target genes. In one mechanism, SS18-SSX forms a bridge that relocates TLE1- aggregated repressors to ATF2-bound loci. The second mechanism involves the modulation of the chromatin remodeling BAF complex by replacing native SS18 with the fusion and ejecting another member, SNF5. Each mechanism has been demonstrated at specific loci in cell lines, but not genome-wide or in tumors. Working from the hypothesis that SS18-SSX expression misregulates the recruitment and/or activity of chromatin remodeling and modifying complexes to create an oncogenic transcriptional profile, the proposed experiments will specifically (1) Determine the necessity of the two described mechanisms to synovial sarcomagenesis, (2) Determine whether SS18-SSX alters BAF function, location, or both, beyond SNF5 ejection, and (3) Determine the drivers of SS18-SSX oncogenesis by defining the genomic locations of the SS18-SSX fusion oncoprotein (and partners) by ChIP-seq, and their impact at occupied loci by RNA-seq. Reverse genetic experiments in an otherwise fully penetrant model of synovial sarcomagenesis in the mouse will test the necessity of central members of each mechanism. Both conditional and temporally inducible expression of SS18-SSX will test the impact of its addition to Snf5-loss induced tumorigenesis in the mouse. Synovial sarcoma will specifically be induced in mice by expression of a novel V5-tagged SS18-SSX fusion, to correct for the difficult interpretation of prior experiments with antibodies non-specific for the fusion, thus enabling genome-wide localization of the fusion and co-localization of partnering proteins at silenced and activated loci. These genomic localization assessments will therefore be performed in an idealized experimental tumorigenesis setting with comparison control assessments available in the tissue most enriched for the pre-transformation cell of origin. Data from this idealized experimental paradigm in the model will then be prioritized and validated with comparative genomics from human synovial sarcoma samples.